Flaviviruses
The flavivirus group (family Flaviviridae) comprises the genera Flavivirus, Pestivirus and Hepacivirus and includes the causative agents of numerous human diseases and a variety of animal diseases which cause significant losses to the livestock industry.
The family Flavivirdae (members of which are referred to herein as flaviviruses) include the genera Flavivirus (e.g. yellow fever virus, dengue viruses, Japanese encephalitis virus and tick-borne encephalitis virus), Pestivirus (e.g. bovine viral diarrhoea virus, classical swine fever virus and border disease virus), Hepacivirus (hepatitis C virus) and currently unclassified members of the Flaviviridae (e.g. GB virus types A, B and C).
The full list of members of the Flaviviridae are defined in detail by the International Committee on Taxonomy of Viruses (the currently accepted taxanomic definition is described in: Virus Taxonomy: The Classification and Nomenclature of Viruses. The Seventh Report of the International Committee on Taxonomy of Viruses (book). M. H. V. van Regenmortel, C. M.
Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle, R. B. Wickner (2000). Virus Taxonomy, VIIth report of the ICTV. Academic Press, San Diego), the contents of which are hereby incorporated by reference.
However, perhaps the most significant flavivirus is the hepatitis C virus (HCV). HCV was first identified in 1989 and it has since become clear that this virus is responsible for most cases of post-transfusion non-A, non-B hepatitis. Indeed, HCV is now recognised as one of the commonest infections causing chronic liver disease and The World Health Organisation estimates that 170 million people are chronically infected. HCV infection results in a chronic infection in 85% of infected patients and approximately 20-30% of these will progress to cirrhosis and end stage liver disease, frequently complicated by hepatocellular carcinoma.
The study of HCV has been hampered by the inability to propagate the virus efficiently in cell culture. However, in the absence of a suitable cell culture system able to support replication of human HCV, BVDV is an accepted cell culture model. HCV and BVDV share a significant degree of local protein homology, a common replication strategy and probably the same subcellular location for viral envelopment.
HCV is an enveloped plus-strand RNA virus belonging to the Flaviviridae family, but classified as a distinct genus Hepacivirus. The HCV genome consists of a single long open reading frame which encodes a ˜3000 amino acid residue polyprotein. This polyprotein is processed co- and post translationally into at least 10 different products including two N-linked glycosylated proteins E1 and E2.
The genome carries at the 5′ and 3′ ends non-translated regions (NTRs) that form stable secondary and tertiary structures. The 5′ NTR carries an internal ribosome entry site (IRES) permitting the direct binding of ribosomes in close proximity to the start codon of the ORF. Thus translation of HCV RNA is mediated by the IRES, rather than the CAP-dependent mechanism typically used by cellular mRNA.
Within the polyprotein, cleavage products are ordered as follows: core (C), envelope protein 1 (E1), E2, p7, non-structural protein 2(NS2), NS3, NS4A, NS4B, NS5A and NS5B. The core protein is a highly basic RNA binding protein forming the major constituent of the nucleocapsid. The envelope proteins E1 and E2 are highly glycosylated type 1 membrane proteins anchored through the carboxy-terminal region. They are embedded into the lipid envelope of the virus particle and associate to form stable heterodimers. The cleavage product p7 is a small hydrophobic peptide of unknown function. The non-structural proteins are involved in viral replication and possess protease (NS2/NS3), helicase (NS3) and RNA polymerase activities (NS5B).
Binding to the host cell probably requires the interaction of E2 or the E1/E2 complex with a receptor that is present on the cell surface.
Due to the lack of an efficient cell culture replication system the understanding of HCV particle assembly is very limited. However, the absence of complex glycans, the localisation of expressed HCV glycoproteins in the endoplasmic reticulum (ER) and the absence of these proteins on the cell surface suggest that initial virion morphogenesis occurs by budding into intracellular vesicles from the ER. Additionally, mature E1-E2 heterodimers do not leave the ER, and ER retention signals have been identified in the C-terminal regions of both E1 and E2. In this case the virus would be exported via the constitutive secretory pathway. In agreement with this assumption, complex N-linked glycans were found on the surface of partially purified virus particles suggesting that the virus transits through the Golgi.
Until recently, interferon-α(IFN-α) was the only therapy with proven benefit for the treatment of HCV infection. Using IFN-αup to 50% of patients show a response to treatment, but this is not sustainable in the majority of patients and there are considerable associated side effects. More recently, greater success has been achieved using IFN-αin combination with the nucleoside analogue ribavirin, but continuing research is required to identify new therapeutic candidates that will have more potent antiviral activity and less severe side-effects.
There is therefore a need for improved anti-flaviviral drugs in general, and anti-HCV drugs in particular.
Glycoproteins and viral development
Glycoproteins are classified into two major classes according to the linkage between sugar and amino acid of the protein. The most common and extensively studied is N-glycosidic linkage between an asparagine of the protein and an N-acetyl-D-glucosamine residue of the oligosaccharide. N-linked oligosaccharides, following attachment to a polypeptide backbone, are processed by a series of specific enzymes in the endoplasmic reticulum (ER) and this processing pathway has been well characterised.
In the ER, α-glucosidase I is responsible for the removal of the terminal α-1,2 glucose residue from the precursor oligosaccharide and α-glucosidase II removes the two remaining α-1,3 linked glucose residues, prior to removal of mannose residues by mannosidases and further processing reactions involving various transferases. These oligosaccharide “trimming” reactions enable glycoproteins to fold correctly and to interact with chaperone proteins such as calnexin and calreticulin for transport through the Golgi apparatus.
Inhibitors of key enzymes in this biosynthetic pathway, particularly those blocking α-glucosidases and α-mannosidase, have been shown to prevent replication of several enveloped viruses. Such inhibitors may act by interfering with the folding of the viral envelope glycoprotein, so preventing the initial virus-host cell interaction or subsequent fusion. They may also prevent viral duplication by preventing the construction of the proper glycoprotein required for the completion of the viral membrane.
For example, it has been reported that the nonspecific glycosylation inhibitors 2-deoxy-D-glucose and β-hydroxy-norvaline inhibit expression of HIV glycoproteins and block the formation of syncytia (Blough et al., Biochemical and Biophysical Research Communications, 141(1), 33-38 (1986)). Viral multiplication of HIV-infected cells treated with these agents is stopped, presumably because of the unavailability of glycoprotein required for viral membrane formation.
In another report, the glycosylation inhibitor 2-deoxy-2-fluoro-D-mannose was found to exhibit antiviral activity against influenza infected cells by preventing the glycosylation of viral membrane protein (McDowell et al., Biochemistry, 24(27), 8145-52 (1985)). This report also studied the antiviral activity of 2-deoxyglucose and 2-deoxy-2-fluoroglucose and found that each inhibits viral protein glycosylation by a different mechanism.
Lu et al (1995) present evidence that N-linked glycosylaton is necessary for hepatitis B virus secretion (Virology 213: 660-665) while Block et al (1994) show that secretion of human hepatitis B virus is inhibited by the imino sugar N-butyldeoxynojirimycin (PNAS 91: 2235-2239). See also WO9929321.
Taylor et al (1988) demonstrate the loss of cytomegalovirus infectivity after treatment with castanospermine or other plant alkaloids and relate this to abberant glycoprotein synthesis (Antiviral Res. 10: 11-26). See also U.S. Pat. No. 5,004,746.
Taylor et al. (1994) show that inhibition of α-glucosidase I of the glycoprotein processing enzymes by 6-0-butanoyl castanospermine has consequences in human immunodeficiency virus-infected T-cells (Antimicrob. Ag. Chemother. 38: 1780-1787) while Sunkara et al (1989) describe anti-HIV activity of castanospermine analogues (Lancet II 1206). See also U.S. Pat. No. 5,004,746.
U.S. Pat. No. 5,385,911 discloses anti-herpes activity in certain castanospermine esters.
However, many other known glycosylation inhibitors have been found to have no antiviral activity. Thus the antiviral activity against enveloped viruses, in general, and the anti-flaviviral activity, specifically, of glycosylation inhibitors is quite unpredictable.
Glucosidase Inhibitors
Castanospermine and certain imino sugars, such as deoxynojirimycin (DNJ), are ER α-glucosidase inhibitors and both potently inhibit the early stages of glycoprotein processing. However, their effects differ substantially depending on the system to which they are applied and they may exhibit quite different specificities, castanospermine being relatively specific for α-glucosidase I.
Castanospermine is an alkaloid, originally isolated from the seeds of Castanospermum australe, having the following formula:

Systematically, this compound can be named in several ways as follows: [1S-(1α, 6β,7α,8β,8αβ)]-octahydro-1,6,7,8-indoli-zinetetrol or [(1S,(1S,6S,7R,8R,8aR)-1,6,7,8-tetrahydroxy-indolizidine or 1,2,4,8-tetradeoxy-1,4,8-nitrilo-L-glycero-D-galacto-octitol. The term “castanospermine” or the first systematic name will be used in the discussion below.
Branza-Nichita et al. (2001) J. Virol 75(8): 3527-3536 show that the Iminosugar N-butyldeoxynojirimycin has an antiviral effect against the Pestivirus B VDV. However, the authors make clear that while treatment with α-glucosidase inhibitors may affect the life cycles of this and other enveloped viruses, it is not possible to generalize to other viruses since the effects may depend crucially on the particular folding pathway used by the viral proteins.
Courageot et al. (2000) J. Virol. 74(1): 564-572 report that the α-glucosidase inhibitors castanospermine and DNJ reduce dengue virus production in an in vitro mouse model. However, no substantial difference in activity between the imino sugar inhibitor DNJ and castanospermine was reported.
WO 99/29321 discloses the use of α-glucosidase inhibitors generally (and imino sugars in particular) in the treatment of inter alia HCV infections. However, no reference is made to castanospermine (or esters or derivatives thereof) specifically in this respect. Instead, the document focuses on the activities of various imino sugars.
Choukhi et al. (1998) J. Virol. 72(5): 3851-3858 report the effect of castanospermine on the interactions between HCV glycoproteins and their chaperones. Castanospermine did not abolish the interaction between HCV glycoproteins and the chaperones calnexin and calreticulin. Rather, castanospermine actually increased the binding of the glycoproteins to calreticulin. The authors suggest that HCV glycoprotein processing may not be sensitive to inhibitors of glycoprotein trimming (such as castanospermine), concluding that:                . . . binding of HCV glycoproteins to and release from calnexin or calreticulin could be independent of trimming . . . of the N-linked glycans.        [Choukhi et al., page 3856, column 1]        
Despite such contra-teachings, the present inventors have now surprisingly discovered that certain esters of castanospermine do in fact exhibit antiviral activity against members of the Flaviviridae (including HCV). Moreover, they have found that the therapeutic index is unexpectedly far superior to that exhibited by other α-glucosidase inhibitors of the imino sugar class (the esters exhibit relatively high antiviral activity and relatively low toxicity). Without wishing to be bound by any theory, it is postulated that these unexpected properties of the castanospermine esters may reflect their relative specificity for a particular class of glycoprotein processing enzymes (viz. α-glucosidase I).